The general aim of the current proposal is to describe, at the molecular level, bile acid 7alpha-dehydroxylation in gut bacteria. Approximately 20 to 25% of the biliary bile acid pool in man is deoxycholic acid, which is a secondary bile acid generated from cholic acid by gut bacteria. The hydrophobic secondary bile acids (deoxycholic and lithocholic acid) are powerful regulators of hepatic cholesterol and bile acid biosynthesis, and are very toxic to mammalian cells. The specific aims of this proposal are to "overexpress" in E. coli two specific bile acid inducible genes from a 7alpha-dehydroxylating Eubacterium species. These genes code for proteins of 58 Kd and 27 Kd polypeptides. We will test the hypothesis that the 58 Kd and 27 Kd polypeptides code for a bile acid ligase (CoA) and 3alpha-hydroxysteroid dehydrogenase, respectively. The final aim is to purify, characterize, clone and sequence a bile acid 5alpha- and 5beta- reductase from Eubacterium species.